PROJECT SUMMARY/ABSTRACT There is a fundamental gap in understanding how to activate and increase tumoricidal activity low-affinity tumor infiltrating lymphocytes (TILs) that recognize antigens derived from dysregulated unmutated proteins. Continued existence of this gap represents an important problem because there are many tumors with a low mutation load that do not respond to current treatments, but elicit low-affinity T cell responses. Our long-term goal is to identify unique inhibitory receptors that are functionally enriched in TILs and epigenetic targets to help establish mechanisms that contribute to development of future therapies for these cancers. The overarching goal of this research is to elucidate pathways that can be harnessed to improve antitumor T cell activity, particularly the activity of low-affinity TILs. The central hypothesis is that the antitumor activity of low- affinity T cells is sensitive to enhancement by blocking inhibitory receptors and targeting epigenetic regulatory regions. This hypothesis has been formulated on the basis of preliminary data produced in the applicants' laboratories. The rationale for the proposed research is that understanding the activation and differentiation of low-affinity T cells has the potential to translate into better understanding of better therapies for cancer that now afflicts one of every two to three people in this country. Guided by strong preliminary data, this hypothesis will be tested by pursuing two specific aims: 1) Establish mechanisms that modulate antitumor activity of low- affinity TILs by defining their inhibitory receptor repertoire, and 2) Establish mechanisms of epigenetic programming in low-affinity TILs. Both of these aims will use retrogenic technology to generate large pools of either high or low-affinity tumor-specific T cells. In the first aim, inhibitory receptors will be identified from a list of ITIM-containing genes that were found by the investigators to be upregulated in TILs and using biochemical approaches that identifies inhibitory receptors from low-affinity T cells using SH2 domains. The genes of interest identified in this aim will be confirmed in other systems and probed on human colorectal samples to determine their expression on human TILs. In the second aim, gene expression and chromatin accessibility of low and high affinity T cells in the periphery and tumor will be examined. The effects of T cells development in tumor antigen-deficient mice will also be examined. The approach is innovative, in the applicant's opinion, because it departs from the status quo of looking at the highest affinity reactions against tumors using novel methods. The contribution of the proposed research will be significant because identification of inhibitory receptors and epigenetic targets expected to activate endogenous low-affinity T cells may have fewer toxicities and activate T cells in some tumors that have not yet experienced the benefits of immunotherapies. Ultimately, such knowledge has the potential to inform the development of immunotherapies for cancers that do not as yet have treatments.